Project Summary Zika virus (ZIKV) is a recently recognized global public health threat due to the wide geographic distribution of risk and potential for severe consequences of congenital infection. There is an urgent need for effective therapeutic strategies against ZIKV that have established safety in humans, especially during pregnancy. Our preliminary data show that chloroquine, hydroxychloroquine and amodiaquine block ZIKV replication in cultured cells at clinically achievable concentrations. These medications have been used for many decades in humans, including during pregnancy. The identification of a class of drugs that are efficacious for ZIKV infection, well tolerated, safe in pregnancy, inexpensive, widely available and orally bioavailable would have the immediately translatable potential to significantly impact this ongoing epidemic. This proposal aims to further develop in vitro models to study the effect of chloroquine and related drugs on ZIKV infection, identify the molecular mechanism(s) of their activity and determine whether these agents demonstrate efficacy in vivo in mouse models. In Aim 1, we will perform dose-response experiments with chloroquine and a panel of related compounds and determine whether protection occurs in primary cells including neural progenitor cells and with currently circulating ZIKV strains. Aim 2 will identify the cellular and molecular mechanism(s) of inhibition by chloroquine and related drugs. We propose that understanding how these drugs work will reveal critical insights into the basic biology of this virus and pathways amenable to therapeutic targeting. In Aim 3, we will determine the efficacy of chloroquine and related compounds in mouse models of ZIKV infection, which may provide preclinical evidence to support clinical trials in humans. The experiments we propose will not only determine whether chloroquine and related drugs are efficacious against ZIKV, but also shed light on the basic biology of this virus, which will inform future drug development. Our findings may be relevant not only to ZIKV, but also related flaviviruses of current medical importance and ones that may emerge in the future.